1. Field of Invention
The present invention relates to protein disulfide isomerase, an enzyme which promotes formation of protein conformation by catalyzing formation of disulfide bonds in a protein, and to a gene thereof. The present invention relates, among the protein disulfide isomerases, to protein disulfide isomerase derived from a strain of methylotrophic yeast, a microorganism suitable for industrial production of valuable proteins due to its high efficiency of expression of heterologous genes and secretion of the expression products, and to a gene thereof.
2. Related Art
Protein disulfide isomerase (PDI) is a major protein present in the lumen of the endoplasmic reticulum (referred to hereinafter as ER) and it was first discovered as having an activity which effects oxidative refolding of a reduced RNase (Goldberger, R. F. et al. (1963) J. Biol. Chem. 238: 628-635). PDI is believed to be an enzyme which catalyzes formation of stable conformation by recombining disulfide bonds of secretory proteins.
It has been pointed out that, in the case of heterologous proteins, in particular secretory proteins, which often have disulfide bonds, recombination of disulfide bonds by PDI as well as protein folding by peptidyl-prolyl-cis-trans isomerase (PPI) represent the rate-limiting step in the secretory process of proteins (Gething, M. J. and Sambrook, J. (1992) Nature 355: 33-45). It has also been demonstrated that PDI promotes folding of proteins consisting of a single domain such as RNase in vitro as well (Jaenicke, R. (1993) Curr. Opin. Struct. Biol. 3: 104-102).
On the other hand, because strain of methylotrophic yeasts grow using methanol as the sole carbon source and they have high yields of cells, they have been used for the production of materials for use in the synthetic chemical industry including, for example, aldehydes such as formaldehyde, epoxides, methylethylketone, and formic acid. Research has been conducted on the possible utilization of the cells per se as a protein source, and the utilization for production of cell components such as amino acids, vitamins, and the like, and some have been put into practical use. In recent years, furthermore, an expression system of heterologous genes using strain of methylotrophic yeasts as the host has been developed and it has been shown that said system has a higher productivity than Saccharomyces yeasts (Japanese Unexamined Patent Publication (Kokai) No. 5-344895).
Its productivity is high especially for secretory proteins. For example, the productivity of glucoamylase derived from filamentous fungi of the genus Rhizopus was 3.4 g/l, which is about 10 times higher than the productivity by Saccharomyces yeasts (Sakai, Y., et al. (1996) Biochim. Biophys. Acta 1308: 81-87). As the strain of methylotrophic yeast, there are known Candida boidinii, Pichia pastoris, Hansenula polymorpha, and the like.
When heterologous proteins are produced by secretory production using recombinant DNA technology, the efficiency of the secretion is thought to be increased by enhancing the speed of folding proteins. Based on such an idea, an example has been disclosed in which the amount secreted of human albumin was increased by about 60% on the average by coexpressing a human PDI gene with the desired gene in a Saccharomyces yeast (Japanese Unexamined Patent Publication (Kokai) No. 5-38771).
Formation or exchange of disulfide bonds which are necessary for appropriate folding of proteins requires environments suitable therefor. For that purpose, eukaryotic cells have intracellular compartments such as the ER or the Golgi apparatus, etc. While passing through the compartments, secretory proteins are subjected to suitable folding or addition of sugar chains and then are secreted out of the cell by means of exocytosis. Many of the secretory proteins of eukaryotic origin have intramolecular disulfide bonds, and formation and exchange of these disulfide bonds taking place in the ER are essential for formation of protein conformation and its secretion.
Accordingly, the PDI which catalyzes reactions for formation and/or exchange of disulfide bonds must be localized or stay in the ER. For this purpose the PDI has a unique amino acid sequence called an ER retention signal sequence at the C-terminal. As ER retention signal sequences there are known Lys-Asp-Glu-Leu (SEQ ID No. 2) for animals and His-Asp-Glu-Leu (SEQ ID No. 3) for Saccharomyces yeasts. When the human PDI gene as described above was expressed in a Saccharomyces yeast, the ER retention signal sequence of the human PDI did not fully function, which was possibly due to inadequate localization of the PDI in the ER. Thus, it is believed that even the highly expressed PDI gene did not cause enhancement in the PDI activity commensurate with the expression in the ER, and accordingly the increment of the amount secreted of the coexpressed secretory protein remained at a value of 60%.
In order for the PDI expressed in a strain of methylotrophic yeast to fully perform its functions, it is preferred to use the PDI derived from a strain of methylotrophic yeast. The reason why the strain of methylotrophic yeast has a high ability of secreting protein as described above is that recombination of disulfide bonds by the PDI which is the rate-limiting step of the protein secretion process takes place efficiently and that the PDI derived from the strain of methylotrophic yeast has a higher specific activity than the PDI derived from other sources or has a higher activity in the ER. However, the PDI of the strain of methylotrophic yeast or the gene thereof was unknown. Accordingly, no studies had been carried out on enhancement of productivity in the expression system of the strain of methylotrophic yeast by using the above PDI or the gene thereof.